Proc. Nati. Acad. Sci. U.SA Vol. 74, No. 5, pp. 2054-20581, May 1977 Cell Biology

Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells* (thyroid hormone/somatotropin/translation in cell-free system/mechanism of hormone action) HISAO SEOt, GILBERT VASSARTt, HUGETTE BROCASt, AND SAMUEL REFETOFFt t Thyroid Study Unit, University of Chicago, School of Medicine, Chicago, Illinois 60637; and * Institute de Recherche Interdisciplinaire, School of Medicine, Free University of Brussels, 1000 Brussels, Belgium Communicated by Elwood V. Jensen, March 10, 1977

ABSTRACT In a cell-free protein-synthesizing system from the T3-stimulated GH but not prolactin synthesis is accompa- a rabbit reticulocyte lysate, total RNA extracted from cultured nied by a specific increase in GH mRNA but not prolactin rat pituitary tumor (GH3) cells directed, in a dose-related man- mRNA activity. Thus, the known effect of T3 on GH production ner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. may predominantly involve a selective stimulation of tran- A marked decrease in growth hormone secretion and growth scriptional activity. hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of tri- METHODS iodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone Cell Culture. Rat pituitary tumor cells originating from the had no effect on prolactin secretion or prolactin mRNA activity. GH3 line were a gift from A. H. Tashjian. This line, derived These data suggest that thyroid hormone may stimulate syn- and thesis of growth hormone throug induction of transcriptional from a single MtTW5 rat pituitary tumor cell, synthesizes activity. The possibility of an additional effect at the posttran- secretes both GH and PRL (10). Except for the medium used, scriptional level has not been excluded. Although thyroid hor- the cells were propagated in culture as previously described mone is believed to have a general effect on a variety of meta- (11). Cells were maintained in modified Eagle's medium bolic processes, some effects, at the molecular level, may be (MEM) containing 10% fetal calf serum. For the experiments, quite selective, as indicated by the observed changes in growth cells were grown in MEM supplemented with 10% serum from hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of in- either normal (N) or thyroidectomized (Tx) rats, with or without dependence from secondary hormonal effects caused by hy- added T3. Each rat received 100,uCi of Na131I on the second pothyroidism and because simultaneous measurement of pro- day following surgical thyroidectomy and serum was harvested lactin mRNA activity serves as a unique internal control. 4 weeks later. Concentrations of endogenous thyroxine and T3 in the normal rat serum were 5.4 ,ug/100 ml and 60 ng/100 ml, The demonstration of triiodothyronine (T3) binding to nuclear respectively, and in the Tx rat serum were 0.6 ,g/100 ml and proteins raises the possibility that thyroid hormone may regulate less than 20 ng/100 ml, respectively. gene expression. Earlier work from Tata's laboratory showed RNA Extraction. Each RNA preparation was obtained from that thyroid hormone-induced protein synthesis was preceded 5 to 15 petri dishes or 30 to 70 X 106 cells treated in the same by formation of new RNA (1). Later, DeGroot et al. and Dil- manner. The medium was removed and saved for hormone man et al. showed increase in the poly(A)-rich fraction of RNA determination. The cells adhering to the bottom of the petri (2, 3). Demonstrations of stimulation of a specific mRNA by dishes were washed once with 3 ml of MEM, then harvested by thyroid hormones were recently provided by Kurtz et al. (4) gentle resuspension in the same medium, using a pasteur pi- and by Roy et al. (5) for a2u-globulin in the rat. However, be- pette. A 1 ml aliquot of the pooled 10 ml cell suspension was cause a number of hormones are known to stimulate the syn- saved for total RNA (12) and DNA (13) determination and the thesis of this protein (6) and because thyroid hormone pro- remainder was centrifuged for 10 min at 600 X g. The cell foundly alters the level of such hormones (7), experiments done pellet was resuspended in 1 ml of buffer containing 25 mM in the whole animal do not provide sufficient evidence for the MgCl2/50 mM KCI/200 mM sucrose/200 mM Tris-HCl, pH direct induction of -d2u-globulin mRNA by thyroid hormone. 8.5. In a rapid succession 7 ml of buffer containing 1% sodium Samuels et al. have reported a quantitative correlation be- dodecyl sulfate/100 mM NaCl/5 mM EDTA/0.02% hepa- tween nuclear T3 receptor occupancy and stimulation of growth rin/10 mM Tris, pH 8.5, was added, followed by 8 ml of phe- hormone (GH, somatotropin) synthesis in cultured rat pituitary nol/chloroform (1:1 vol/vol) presaturated with a buffer con- cells (8). We have adopted a similar experimental model to taining 100 mM NaCl/1 mM EDTA/10 mM Tris. Extraction study more directly the action of thyroid hormone on the in- was carried out by manual agitation for 5 min. Following duction of a specific mRNA. In a recent report, we have shown centrifugation for 10 min at 9000 X g the aqueous phase was that GH and prolactin mRNA activities in the total RNA ex- removed, and the organic phase containing the denatured tracted from a rat pituitary cell line (GH3) that actively syn- protein interphase was re-extracted after addition of 8 ml of the thesizes both hormones can be quantitated using a rabbit re- suspension buffer. The two aqueous phases were pooled and ticulocyte lysate cell-free system (9). In this report, we show that extracted four consecutive times with an equal volume of phenol/chloroform. During the final extraction no denatured Abbreviations: T3, triiodothyronine; GH, growth hormone (somato- protein interphase was present. Residual phenol in the aqueous tropin); Tx, thyroidectomized; N, normal; NRS, normal rabbit serum; phase was removed by a single extraction with chloroform, NMS, normal monkey serum; MEM, modified Eagle's medium. was added to a final concentration of 2 * Presented in part at the Fifty-Second Meeting of the American following which LiCl Thyroid Association, Inc., Toronto, Canada, September 15-18, M. The RNA was allowed to precipitate at 4° overnight. After 1976. centrifugation for 10 min at 9000 X g the RNA pellet was 2054 Downloaded by guest on September 26, 2021 Cell Biology: Seo, et al. Proc. Nati. Acad. Sci. USA 74 (1977) 2055

Table 1. Radioactivity in O-RNA and in immunologic blanks Radioactivity RNA, Specific antiserum Normal Anti-IgG serum in precipitate, Aug/ml (1st antibody)* carrier serumt (2nd antibody)t mean cpm ± range 0 Monkey anti-rat GH Goat anti-monkey IgG 1813 ± 187 o NMS Goat anti-monkey IgG 1953 ± 17 200 NMS Goat anti-monkey IgG 1933 ± 33 0 Rabbit anti-rat prolactin Goat anti-rabbit IgG 913 ± 13 0 NRS Goat anti-rabbit IgG 1058 ± 167 200 NRS Goat anti-rabbit IgG 960 ± 16

For details in preparation, see Methods. Volume and dilution of sera added to 200 or 250 ,. oflysate are given in the footnotes. Acid-precipitable radioactivity was 16.76 X 106 cpm. * Monkey anti-rat GH: 5 Al (1:50); rabbit anti-rat prolactin: 5 pl (1:25). t NMS = normal monkey serum: 5 Ml (1:50); NRS = normal rabbit serum: 5 Ml (1:5). 1 Goat anti-monkey IgG: 25 Al (undiluted); goat anti-rabbit IgG: 5 Al (undiluted).

washed two times with cold 2 M LiCl followed by a single wash out on separate aliquots of lysate or on the same lysate in se- with 66% (vol/vol) ethanol in 0.3 M NaCl. The RNA was dis- quence. Either method achieved excellent recovery and re- solved in water and absorbance was read at 260 and 280 nm. producibility (Table 2). The specificity of the antibodies was The ratio in all preparations ranged from 1.9 to 2.1. The amount evaluated by their ability to precipitate purified authentic rat of RNA was calculated assuming that 1 A260 = 40,ug. After an GH and prolactin labeled with 125I. The antiprolactin serum ethanol precipitation, the final RNA preparation was dissolved was specific, and cross reaction of the anti-GH serum with in water to the desired concentretion to be used for transla- prolactin was eliminated by addition of 1 ,ug of prolactin tion. without affecting its reactivity with GH (Fig. 1). Furthermore, Cell-Free Protein Synthesis. Translation was carried out in as previously shown by comigration of the immunoprecipitates a rabbit reticulocyte lysate cell-free system as described by of authentic hormones and cell-free translated material on so- Palmiter et al. (14). After incubation for 1 hr at room temper- dium dodecyl sulfate/polyacrylamide gel electrophoresis (9), ature with RNA and [3H]leucine (Amersham, 53 Ci/mmol) at 95% of the radioactivity appeared as sharp single peaks. In 170 MCi/ml, the reaction was stopped by the addition of 0.1 accordance with findings by Sussman et al. (17), Evans et al. ml/ml of reaction mixture of a solution containing the following (18), and Maurer et al. (19), the hormones translated in the additives and bringing their concentrations to 0.02 M sodium cell-free system were of slightly larger molecular weights than phosphate buffer and 0.15 M NaCl, 1% (vol/vol) Triton X-100, the authentic hormones. 1% sodium deoxycholate, and 0.10% unlabeled L-leucine, pH Quantitation of GH and Prolactin Concentration in Cul- 7.5. The mixture was then centrifuged for 60 min at 100,000 ture Media. GH and prolactin concentrations in the cell culture X g in a Beckman rotor SW 60. The supernatant was used for media were determined by radioimmunoassays as previously trichloroacetic acid and immunoprecipitations described below. described (11). All media were changed 48 hr prior to termi- Blank reaction mixtures contained all additives except for RNA, nation of the experiments. Thus, concentration of hormones which was substituted by an equal volume of water. represent the cumulative secretion by the cells into the medium Trichloroacetic Acid Precipitation. Five microliter aliquots over 48 hr. The cells do not store significant amounts of hor- of the centrifuged lysate were spotted onto Whatman GF/A mone (11) and the rate of hormonal degradation has not been filter disks and processed as described by Schimke et al. determined. (15). Immunoprecipitation. GH and prolactin synthesis in the Table 2. Recovery with single and sequential lysate was measured by double antibody precipitation. Anti- immunoprecipitations bodies against rat GH and rat prolactin were a gift from A. Parlow and second antibodies, anti-rabbit and anti-monkey IgG, 1st immunoprecipitation 2nd immunoprecipitation were from V. S. Fang. Duplicate aliquots containing 200-250 Al of the lysate were incubated with 5,l of diluted first antibody Specific Specific cpm* Specific Specific cpm* for 1 hr at room temperature, and for 16 hr at 40, followed by antiserum in precipitate antiserum in precipitate incubation for 30 min at room temperature and for 8 hr at 40 with the appropriate second antibody (see legend to Table 1). Anti- The precipitate was centrifuged, washed with 1 ml of phos- prolactin 16,815 + 30 Anti-GH 8,479 ± 90 phate-buffered saline, resuspended, and cleaned by centrifu- Anti- Anti- prolactin 16,815 ± 30 prolactin 393 ± 30 gation in Microfuge tubes through 1 M sucrose as described by Anti-GH 7,514 ± 332 Rhoads et al. (16) and dissolved in 1 ml NCS (Amersham) prior to counting. The radioactivity in lysates incubated without Lysate was programmed with 200 Mug of RNA per ml of reaction added RNA (0-RNA blanks) and in immunoprecipitates in the mixture. Acid-precipitable radioactivity was 24.93 X 106 and 26.58 presence of normal rabbit or monkey sera, rather than the X 106 cpm in lysates with or without added RNA, respectively. Im- specific antisera (immunologic blanks) was similar and con- munologic blanks on the 1st and 2nd immunoprecipitation had, re- stituted from 0.004 to 0.020% of the acid-precipitable radio- spectively, 954 I 17 and 978 ± 45 cpm with NRS and 2223 ± 201 and 1915 I 232 cpm with NMS. 0-RNA blanks precipitated with anti- activity (Table 1). Specific immunoprecipitable radioactivity prolactin and anti-GH serum had 1461 + 67 and 2754 1 46 cpm, re- was calculated by subtraction of the appropriate blank. Pre- spectively. All data are expressed as mean ± range. cipitation with antiprolactin and anti-GH serum was carried * cpm in immunoprecipitates minus blanks. Downloaded by guest on September 26, 2021 2056 Cell Biology: Seo, et al. Proc. Nati. Acad. Sci. USA 74 (1977)

_ x 103 125l-Prolactin 9- 6 14- 100r I 7 V r-12- C, 801

- C

D 60 0 8- .SCu Q 6 O 40 E a 4- E 0 201 E E 2- E L. L- C3 .O 500 NRS Unlabeled 0 500 L6101 000 0 0- prolactin, NMS anti-Prolactin ng/tube Total RNA, pg/ml FIG. 2. Relation of RNA input to cell-free synthesis of prolactin anti-GH and GH. Total RNA extracted from cultured rat pituitary tumor cells FIG. 1. Specificity of antisera to rat prolactin and GH. Antisera was added to rabbit reticulocyte lysate cell-free protein-synthesizing were incubated with 125I-labeled purified rat prolactin (1251-prolactin) system to yield the final concentration indicated in the abscissa. or GH (1251-GH) under the standard reaction conditions described in the Methods section. Antiprolactin reacted only with 125I-prolactin. The amount of precipitable 125I-GH was similar to the NRS blank. were no changes in prolactin secretion. A corresponding de- In the absence of unlabeled prolactin, anti-GH reacted equally with crease in GH mRNA activity was observed in cells grown in 125I-GH and 1251-prolactin. Addition of 1000 ng of unlabeled prolactin media supplemented with thyroid hormone-poor rat serum. reduced the immunoprecipitable 1251-prolactin to the level of the Addition of T3 to the Tx rat serum prevented the decrease in NMS blank without affecting the precipitability of 125I-GH. For ab- GH mRNA activity. The total amount of RNA per cell was not breviations see legend to Table 1. affected by the conditions of culture. RNA to DNA ratios ex- RESULTS pressed as mean ± range in two experiments were as follows: 2.4 ± 0.3 for control cells grown in normal rat serum, 2.5 + 0.3 Application of the Reticulocyte Lysate System to Quan- for cells grown in Tx serum, and 2.7 + 0.2 for cells grown in Tx titate GH and Prolactin mRNA Activity. We have previously serum supplemented with T3. shown a linear dose response between the radioactivity in GH To eliminate the possibility that decrease in GH mRNA ac- immunoprecipitates and the input of RNA extracted from rat tivity observed in cells grown in Tx rat serum was due to inhi- pituitary tumors transplanted in vvo. The linear dose response bition of mRNA translation in the cell-free system, the relation extended over the range of 10-300 Asg of RNA per ml of lysate of RNA input to GH synthesis in the lysate was studied. As (9). Results from a similar study conducted with total RNA shown in Fig. 4, the proportionality between RNA input and extracted from the same cells grown in culture are shown in Fig. GH synthesis was preserved in the three RNA preparations. 2. There was a dose-dependent increase in both prolactin and GH immunoprecipitable radioactivity. Depending upon the GH and prolactin secretion into medium GH mRNA activity lysate preparation used, 50-60% of the total radioactivity was acid-precipitable in the absence of exogenous RNA. With the GH addition of increasing amounts of RNA, and depending upon = Prolactin the lysate preparation, the acid-precipitable radioactivity T jLIn r-4, AI - 200 usually but not always declined to a minimum of 25% when the m 10 amount of 200 of RNA per ml of reticulocyte lysate largest ,g x was added. Depending upon the source and quantity of RNA a 160' E 4- added, 0.023-0.210% of the acid-precipitable radioactivity was z a CDa immunoprecipitable with anti-GH or antiprolactin sera. Im- m 120 n 3- and 0-RNA blanks had 0.004-0.020% of the acid- 0i munologic cmC O precipitable radioactivity. C 80 2- Effect of Thyroid Hormone Deprivation on the Secretion 0a c, and mRNA Activity of GH. In this study, cells plated in MEM ~0 C I- serum were three groups of co E containing fetal calf divided into 0 E 15 plates. Medium in the first group was replaced with MEM 0 b. v- O0 supplemented with 10% normal rat serum; in the second group, N Tx N Tx with MEM containing 10% Tx rat serum; and in the third group the 10% Tx rat serum added to MEM was enriched with 80 ng TS of T3 per 100 ml to bring thyroid hormone concentration to a FIG. 3. Effect ofthyroid hormone on GH and prolactin synthesis physiologic level. Following 6 days incubation in these condi- and on the GH mRNA activity. Rat pituitary tumor cells were cul- media were cells were harvested, RNA was tured in media supplemented with 10% of thyroidectomized rat serum tions, collected, (Tx), normal rat serum (N), or thyroidectomized rat serum with added extracted, and mRNA activity was measured. Results are shown T3 (Tx + T3). Endogenous GH and prolactin synthesis, estimated in Fig. 3. Culture in conditions of thyroid hormone deficiency from the amount secreted into the medium, and the GH mRNA ac- profoundly depressed GH secretion. This was prevented by the tivity were measured. Data are expressed as mean i range ofduplicate addition of T3 to the thyroid hormone-deficient serum. There determinations. For methodologic details see text. Downloaded by guest on September 26, 2021 Cell Biology: Seo et al. Proc. Natl. Acad. Sci. USA 74 (1977) 2057

+T3 (80ng/dO) Table 3. Effect of T3 on GH and prolactin secretion and on mRNA activity E 0 N Ratio, mean ± range 3-

a)IV Tx Tx + T3

la Hormone Measurement N N 0 GH C Secretion, E ng/gg of DNA per 48 hr 0.24 ± 0.16 0.82 ± 0.37 mRNA ac- tivity, 0 100 2'00 cpm in im- Total RNA, jig /ml munopre- FIG. 4. Immunoreactive GH in lysates programmed with in- cipitate 0.062 ± 0.021 0.84 ± 0.132 creasing amounts of RNA derived from cells cultured under various Prolactin Secretion, conditions. For abbreviations see legend to Fig. 3. Methodological ng/gg of details appear in the text. and translation Culture, treatment, assays DNA per using the same reticulocyte lysate were carried out simultaneously. 48 hr 1.2 ± 0.05 1.4 ± 0.34 mRNA activ- Also, there was no evidence for inhibition of endogenous he- ity, moglobin synthesis by the lysate, because acid-precipitable cpm in im- radioactivity was identical in all reactions with same RNA munopre- input, irrespective of its origin. Thus, quantitation of the mRNA cipitate 1.4 ± 0.08 1.3 ± 0.13 activity most likely reflects mRNA content. Reversal of the Effects of Thyroid Hormone Deprivation For abbreviations and experimental design, see text. by Addition of T3. In this experiment, cells were allowed to globulin is subject to influences by a variety of hormones of grow for 8 days in MEM containing 10% Tx rat serum, fol- ponthyroidal origin, such as growth hormone (6), the level of lowing which 80 ng of T3 per 100 ml of Tx rat serum was added which is known to be profoundly affected by thyroid hormone and culture was 2 more were continued for days. Control plates (7). grown in the presence of either 10% normal rat serum or Tx rat In the present work we have shown that thyroid hormone serum for the periods of 8 and 10 days. Results of GH and deprivation decreases both the synthesis and mRNA activity prolactin secretion into the media and the respective mRNA of GH in rat pituitary cells grown in culture. The effect was not activities are shown in Table 3, and are for clarity expressed as due to other metabolic alterations caused by hypothyroidism, the ratio of Tx/normal or Tx + T3/normal. As compared to because addition of thyroid hormone to the medium containing normal, a 4-fold depression in GH secretion and 16-fold de- hypothyroid rat serum prevented the occurrence of the changes crease in GH mRNA activity was found in cells grown in Tx rat seen when hypothyroid rat serum was used alone (Fig. 3). The serum for the entire period of incubation. Culture for 2 days effect could be completely reversed within 48 hr following the after addition of T3 was sufficient to normalize both GH se- addition to the medium of physiologic concentrations of T3. In cretion and GH mRNA activity. Irrespective of hormonal preliminary experiments, induction of synthesis and mRNA treatment, there were no significant changes in prolactin se- activity of GH was observed as early as 6 hr after addition of cretion or prolactin mRNA activity. T3 to cells cultured in Tx rat serum. Furthermore, it appears that the effect of thyroid hormone is selective on GH, because DISCUSSION in the same cell preparations no changes occurred in prolactin Thyroid hormone is known to affect a variety of metabolic synthesis or mRNA activity (Table 3). Data presented in ab- processes. While these effects are well recognized, the mech- stract form by Martial et al. (26) are in agreeme..t with our anism by which thyroid hormone influences these processes has observation of T3-induced GH mRNA activity, in another cell remained largely a matter of speculation (20). The recent line of rat pituitary tumors. Because this cell line possesses T3 demonstration of a specific nuclear T3 receptor in various tissues nuclear receptors and their ability to synthesize GH is quanti- (21-23), coupled with work indicating that treatment with tatively related to nuclear T3-receptor occupancy (8), the thyroid hormone increases polymerase (24) or chromatin demonstration of selective induction of GH mRNA suggests that template activity (25) and the poly(A)-rich fraction of RNA (2, expression of thyroid hormone action requires the following 3), suggests that thyroid hormone action may involve changes sequence of events: hormone penetration within the cell, at the transcriptional level. The demonstration that the hormone binding to nuclear receptor proteins, induction of transcrip- induces specific mRNA species, coding for proteins known to tional activity, synthesis of specific protein, and finally the be under hormonal regulation, would show how specificity is metabolic effects induced by the latter. conferred and provide a more direct proof for the mechanism Although, in all experiments, the thyroid hormone-induced of thyroid horomone action. Such evidence appears to have changes in the amount of GH were always associated with been provided by Kurtz et al. (4) and by Roy et al. (5), who corresponding changes in GH mRNA activity, direct propor- showed that treatment of male hypothyroid rats for 7-8 days tionality between medium GH and cell GH mRNA activity with pharmacologic doses of thyroxine restored to normal both could not always be obtained. For example, while data pre- urinary excretion of and mRNA activity for the hepatic protein sented in Fig. 3 show an average of 7.5-fold decrease in both a2u-globulin. However, the interpretation of these experiments GH synthesis and mRNA activity with thyroid hormone de- remains complicated by the fact that the synthesis of a2u- privation, in Table 3, under similar circumstances, an average Downloaded by guest on September 26, 2021 2058 Cell Biology: Seo et al. Proc. Natl. Acad. Sci. USA 74 (1977) of 4-fold decrease in GH synthesis was associated with a 16-fold 1. Tata, J. R. (1969) Gen. Comp. Endocrinol. 2,385-397. decrease in GH mRNA activity. While this discrepancy may 2. DeGroot, L. J. & Rue, P. A. (1976) Fifth International Congress be methodologic in origin, the possibility of an additional effect of Endocrinology, Hamburg, Germany, Abstr. no. 578. of thyroid hormone at the posttranscriptional level cannot be 3. Dillman, W. H., Mendecki, J., Koerner, D. W. & Oppenheimer, J. H. (1976) Fifth International Congress of Endocrinology, excluded (27-29). The following methodologic problems should Hamburg, Germany, Abstr. no. 581. be considered. The endogenous synthesis of GH and prolactin 4. Kurtz, D. T., Sippel, A. S. & Feigelson, P. (1976) Biochemistry was estimated from secretion rate over 48 hr, in turn calculated 15, 1031-1036. from the hormonal concentration in the medium and cell 5. Roy, A. K., Schiop, M. J. & Dowbenko, D. J. (1976) FEBS Lett. number at the termination of the experiment. Rates of cell di- 64,396-399. vision and GH secretion may vary over 48 hr under different 6. Roy, A. K. (1973) J. Endocrinol. 56,295-301. culture conditions. The rate of hormonal degradation has not 7. Hervas, F., Morreale de Escobar, G. & Escobar del Rey, F. (1975) been taken into account. On the other hand, although the in- Endocrinology 97,91-101. tracellular hormone content was not measured, at 48 hr it 8. Samuels, H. H., Shapiro, L. E. & Tsai, J. S. (1976) 58th Annual meeting of the Endocrine Society, San Francisco, California, represents less than 3% of the medium content. From the point Abstr. no. 266. of view of mRNA quantitation, the turnover rate of the mes- 9. Brocas, H., Seo, H., Refetoff, S. & Vassart, G. (1976) FEBS Lett. senger and the residence time of T3 at the receptor site should 70, 175-179. be considered. Acute induction of thyroid hormone deprivation 10. Tashjian, A. H., Jr., Bancroft, F. C. & Levine, L. (1970) J. Cell causes a very gradual decline in GH mRNA activity over at least Biol. 47,61-70. 6 and possibly 10 days. Furthermore, it is practically impossible 11. Seo, H., Refetoff, S. & Fang, V. S. (1977) Endocrinology 100, to prepare a serum totally devoid of thyroid hormone. Attempts 216-226. to extract the hormone modify the serum composition in a va- 12. Munro, M. N. & Fleck, A. D. (1966) in Methods ofBiochemical riety of other substances and serumless media are inadequate Analysis, ed. Glick, D. (Interscience Publishers, New York), Vol. for the of culture 2 that is nec- 14, pp. 113-176. period extending beyond days 13. Burton, K. (1956) Biochem. J. 62, 315-323. essary to induce a state of thyroid hormone deprivation. Thus, 14. Palmiter, R. D., Oka, T. & Schimke, R. T. (1973) J. Biol. Chem. under the present experimental conditions, it is impossible to 248,2031-2039. determine whether thyroid hormone may not indeed switch 15. Schimke, R. T., Rhoads, R. E. & McKnight, G. S. (1974) in de novo the GH gene. We have eliminated the possibility of Methods in Enzymology, eds. Moldave, K. & Grossman, L. inhibition of mRNA translation in the cell-free system by (Academic Press, New York), Vol. 30, pp. 694-701. showing a good dose response with various RNA preparations, 16. Rhoads, R. E., McKnight, G. S. & Schimke, R. T. (1973) J. Biol. and the preservation of prolactin mRNA activity in preparations Chem. 248, 2031-2039. with virtually no GH mRNA activity. In fact, the former serves 17. Sussman, P. M., Tushinski, R. J. & Bancroft, F. C. (1976) Proc. as a unique internal control for the relative activity of the Natl. Acad. Sci. USA 73,29-33. 18. Evans, G. A. & Rosenfeld, M. G. (1976) J. Biol. Chem. 251, cell-free protein-synthesizing system, for possible errors from 2842-2847. extraneous factors affecting cell-free translation, for mRNA 19. Maurer, R. A., Stone, R. & Gorski, J. (1976) J. Biol. Chem. 251, recovery, and for the relative biologic quality of various RNA 2801-2807. preparations tested. 20. Bernal, J. & Refetoff, S. (1977) Clin. Endocrinol., in press. 21. Oppenheimer, J. H., Schwartz, H. L. & Surks, M. I. (1972) J. Clin. The authors are grateful to Dr. Armen H. Tashjian, Jr. for providing Endocrinol. Metab. 35, 330-33. the GHS cell line, to Dr. Albert Parlow for making available rat GH 22. Samuels, H. H. & Tsai, J. S. (1973) Proc. Natl. Acad. Sci. USA 70, and prolactin antisera, to Dr. Victor S. Fang for the anti-rabbit and 3488-3492. anti-monkey IgG sera, to Dr. Gerald Burke for the T3 antiserum, to 23. DeGroot, L. J., Refetoff, S., Strausser, J. & Barsano, C. (1974) Proc. the National Pituitary Agency for GH and prolactin radioimmunoassay Natl. Acad. Sci. USA 71, 4042-4046. kits, and to Dr. Ruben Matalon for assistance and advice in cell culture 24. Griswold, M. D. & Cohen, P. P. (1972) J. Biol. Chem. 247, methodology. The technical assistance of Mrs. Ofelia Gomez and Mr. 353-359. Swen R. Hagen, and the secretarial help of Mrs. Yolanda W. Richmond 25. Kim, K.-H. & Cohen, P. P. (1966) Biochemistry 55, 1251- are also acknowledged. This work was supported in part by U.S. Public 1255. Health Service Grant AM-15070. G.V. is Charg6 de Recherche at the 26. Martial, J. A., Seeberg, P. H., Goodman, H. M. & Baxter, J. D. Belgian Fonds National de la Recherche Scientifique. His travel ex- (1976) 52nd Meeting of the American Thyroid Association, penses were supported through a generous gift from Mr. and Mrs. Toronto, Canada, Abstr. no. T-10. Harry Katz. 27. Sokoloff, L., Roberts, P. A., Januska, M. M. & Kline, J. E. (1968) The costs of publication of this article were defrayed in part by the Proc. Natl. Acad. Sci. USA 60,652-659. payment of page charges from funds made available to support the 28. Yang, S. S. & Sanadi, D. R. (1969) J. Biol. Chem. 244, 5081- research which is the subject of the article. This article must therefore 5082. be hereby marked "advertisement" in accordance with 18 U. S. C. 29. Mathews, R. W., Oronsky, A. & Haschemeyer, A. E. V. (1973) §1734 solely to indicate this fact. J. Biol. Chem. 248,1329-1333. Downloaded by guest on September 26, 2021